Endotoxic shock is still the main cause of death in hospital. Strategies for combating the effects of endotoxic shock are centred on counteracting the bacterial agents responsible for these effects, restoring the hemodynamic parameters, preventing cellular activation and modifying the action of the defence mechanisms (Boyd O; Current Opinion in Anaesthesiology 1996, 9:98)
It is currently accepted that the inflammatory response to bacterial products directly contributes to endotoxic shock (Parillo J E; New England Journal of Medicine 1993, 328:1471). Toxic bacterial products and those released during tissue damage activate the defence mechanisms, with implication of cells such as neutrophils, monocytes, macrophages and endothelial cells, and of mediators such as cytokines, platelet activation factor, metabolites of arachidonic acid and nitric oxide, leading to hemodynamic changes and organ lesions for the host (Moldawer L L; Critical Care Medicine 1994,22:3). Many cytokines have been proposed as markers of the seriousness of the development of septic shock, The levels of circulating TNF-, IL-1, IL-6 and IL-8 have been correlated with e probability of overcoming a septic episode. TNF-, and IL-1 administrated to humans or experimental animals reproduce many of the hemodynamic signs of septic shock (Tracey K J et al., 1986, Science 234:470). Their inhibition by injection of antagonist receptors and blocking monoclonal antibodies have been studied with a wide range of results (Fisher C J et al.; 1994, Critical Care Medicine, 22:12). Of the immunological markers the levels of circulating IL-6 are the best indicators of the seriousness of the sepsis and the possibilities of overcoming the episode (Liaw Y S et al; 1997, Journal of the Formosan Medical Association, 96:685). Despite the advances in knowledge of the mechanisms and of the technical and pharmacological progress there are still few results in terms of an improvement in the data for mortality rate. This rate corresponds to 200,000 deaths per year in the United States and Europe (Vicent J-L and Chamlou R; Current Opinion in Anaesthesiology 1996, 9:146).
The Vasoactive Intestinal Peptide (VIP) is a basic peptide of 28 amino acid units whose sequence is (Mutt V and Said S I; European Biochemistry 1974, 42:581):
His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2, (SEQ ID NO;1)
It was initially isolated from the small intestine of pig and later identified in the brain and terminals of the peripheral nervous system. It was established to be a neuropeptide with neuromodulating properties (Fahrenkrug J; Pharmacology and Toxicology J. 1993, 72:354). Its name is derived from its peripheral vasodilatory properties. VIP has also been identified in rat mast cells and in granulomas (Cutz E. et al.; Nature 1978, 275:661). Immunological studies carried out on histologic sections of thymus, spleen and lymphatic ganglia from rats have identified immunoactive VIP in the lymphocytes of these organs (Leceta et al. Advances in Neuroimmunology 1996, 6:29). VIP exercises its biological effects through membrane receptors belonging to the super family of seven hydrophobic domains coupled to G proteins, which transduce information to the end effector molecules (Laburthe M y Couvineau A; Annals of the New York Academy of Sciences 1988, 527:296). Receptors for VIP have been characterised in several tissues such as liver and adipose tissue among others. These correspond to two types, the so-called VIP1-R (Ishihara T et al.; Neuron 1992, 8:811) and VIP2-R (Lutz E. et al. FEBS Letters 1993, 334:3). In the immune system specific receptors have been characterised for VIP in a variety of immune cells which include human peripheral lymphocytes, human monocytes, rat and mouse lymphocytes, rat alveolar macrophages and peritoneal macrophages of rat and mouse (Delgado M et al.; Regulatory Peptides 1996, 62:161). VIP modulates a great variety of immune functions such as phagocyte function, at every stage of the process, the proliferative response, production of immunoglobin, NK activity and cytokine production (Ganea et al.; Advances in Neuroimmunology 1996, 6:61).
The pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the family of peptides of secretin/VIP/glucagon, of which two molecular forms are known, namely PACAP-38 AND PACAP-27, whose sequences are as indicated (Ogi K et al., Biochemical and Biophysical Research Communication 1993, 196:1511): PACAP-38
His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Thr-Leu-Ala-Ala-Val-Leu-Gly-Lys-Arg-Tyr-Lys-Arg-Tyr-Lys-Gln-Arg-Val-Lys-Asn-Lys-NH2, (SEQ ID NO:2)
PACAP-27
His-Ser-Asp-Gly-Ile-Phe-Thr-Asp-Ser-Tyr-Ser-Arg-Tyr-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Ala-Ala-Val-Leu-NH2, (SEQ ID NO:3)
Both peptides are widely distributed in the central and peripheral nervous system. There are also PACAP producing cells in lung, pancreatic B cells and intestine (Arimura A; Regulatory Peptides 1992, 37:287). In the immune system a large abundance of PACAP positive cells have been described in central and peripheral lymphoid organs (Gaytan F et al.; Cell and Tissue Research 1994, 276:233). For PACAP three types of receptor have been described (Shivers B D et al,; Endocrinology 1991, 128:3055; Inagaki N y col.; Proceedings of the National Academy of Sciences USA 1994, 91:2679): the PACP type I receptor (PACAP-R-I) with equal affinity for PACAP-38 and PACAP-27, but which has an affinity 300 to 1000 times less for VIP; the PACAP type II receptor (PACAP-R-II) which recognises with the same affinity VIP, PACAP-38 and PACAP-27 and is thus denominated the common receptor of VIP-PACAP and corresponds to the receptor VIP VIP1-R, and the PACAP type III receptor (PACAP-R-III) which corresponds to the receptor VIP VIP2-R. Up until present there have been few studies on the biological actions of PACAP on the immune system. The effects of PACAP are often similar to those of VIP mnodulating the phagocyte function and proliferative responses.
The object of this invention is to develop preparation of VIP, PACAP and analogues thereof as therapeutic agents in the treatment of endotoxic shock.
The treatment consists of the administration to mammals in need thereof, of an effective quantity of an agent that inhibits the production of tumor necrosis factor (TNF) in an acceptable pharmaceutical vehicle.
VIP and PACAP have anti-inflammatory effects and inhibit the production of IL-1, IL-6 and TNF-, in animal models of the induction of endotoxic shock. As these cytokines play an important role in the development of said syndrome, VIP and PACAP can be used to regulate their production.
It is known that most of the effects of the endotoxic shock are mediated by activation of the immune system and the inflammatory mechanisms of the host as response to bacterial products. Macrophages play a key role in this process as after their activation factors such as nitric oxide, prostaglandins and cytokines responsible for symptoms such as fever, hypotension, disseminated micro-coagulation, multiple organ failure and finally death, are produced. Similarly, high levels of circulating TNF, IL-1 and IL-6 associated with endotoxemia have been described. In animal models these symptoms are reproduced both by administration of bacterial endotoxins (LPS) and by injection of TNF and IL-1. Other studies have underlined the diagnostic value in terms of the probability of survival represented by the levels of circulating IL-6.
The tumor necrosis factor (TNF) is produced by several types of cell that include monocytes and macrophages, T and B lymphocytes, neutrophils, mast cells, tumorous cells and fibroblasts. It is an important regulatory factor in other pro-inflammatory cytokines, such as IL-1xcex2, IL-6 and IL-8. TNFxcex1 induces the expression of adhesion molecules in endothelial cells, activates leukocytes to destroy the microorganisms, acts on the hepatocytes to increase the synthesis of serum proteins which contribute to the acute phase response and activate the coagulation system. Overproduction thereof leads to immunopathologic diseases, autoimmunity and inflammation.
IL-6 is a multi-functional cytokine produced both by lymphocytes and by non-lymphoid cells. It regulates several aspects of the immune response, such as the production of proteins that mediate acute phase and hematopoiesis. Furthermore, it acts as a mediator in inflammatory response. Its production is regulated by several factors, which include TNFxcex1, IL-1 and bacterial endotoxin (LPS).
Strategies of neutralisation of these cytokines have been tested in the treatment of endotoxic shock but the results do not indicate that there is a greater long-term survival. A treatment that inhibits the production of TNF and, IL-6 would represent a considerable improvement in the evolution of endotoxic shock and in the probabilities of survival. Administration of VIP and PACAP in animal models achieves these effects and our invention consists of using a treatment with these neuropeptides for increasing the survival in cases of endotoxic shock.